Current evidence suggests that the Cl/HCO3 exchanger and the Cl channel in salivary glands play an important role during stimulation-induced fluid secretion, pHi homeostasis and cell volume regulation. The intracellular anionic milieu acts as the driving force for fluid and electrolyte secretion, and may be essential for efficient secretory granule discharge. Elucidating the structure-function relationships of the Cl transporting pathways associated with fluid and electrolyte secretion may therefore be critical to understanding salivary gland dysfunction caused by a myriad of perturbations (e.g. postradiation and drug-induced xerostomia, Sjogrens disease or chloride transporting defects such as cystic fibrosis). However, our understanding of the mechanisms of anion translocation via these Cl transport proteins is lacking. Two approaches will be used to examine questions concerning the structure-function relationships of anion transporters in salivary glands. 1) We will determine the inhibitor susceptibility and kinetic properties of anion transport proteins in salivary glands. These studies will provide information about transport mechanisms and will reveal possible similarities to anion transport proteins in other tissues. For example, examining either the dependence of the Cl/HCO3 exchange rate on internal and external Cl, or by determining the inhibitor susceptibility of the Cl/HCO3 exchanger, we will disclose the kinetic mechanism(s) by which anion exchange occurs in salivary glands. The use of patch clamp studies will yield the number of channels/cell and Cl current/cell which will aid in defining the detailed role Cl ions play in secretion. 2) We will characterize clones from rat salivary gland cDNA libraries encoding the salivary gland anion exchanger to determine the degree of homology with other anion transport proteins and identify alternate transcription/translation possibilities as have been observed in the kidney and erythrocytes. We will then identify potentially important domains for anion exchange activity and perform site-directed mutagenesis to define domains critical for salivary gland function. We will attempt to isolate clones encoding for the salivary gland Cl channel using probes generated against the cystic fibrosis gene, based upon sequence thought to contain a transmembrane conductance regulator, the putative Cl channel. Insight gained from these studies may therefore aid in developing rationales for preventing and/or treating salivary gland dysfunctions.